{"title":"利用 CRISPR-Cas9 工具在酿酒酵母中表达烟曲霉壳聚糖酶","authors":"Qingshuai Zhang, Hui Cao","doi":"10.1186/s40643-023-00718-4","DOIUrl":null,"url":null,"abstract":"<p>Chitooligosaccharides (COS) find numerous applications due to their exceptional properties. Enzymatic hydrolysis of chitosan by chitosanase is considered an advantageous route for COS production. Heterologous expression of chitosanase holds significant promise, yet studies using commonly employed <i>Escherichia coli</i> and <i>Pichia pastoris</i> strains encounter challenges in subsequent handling and industrial scalability. In this investigation, we opted for using the safe yeast strain Saccharomyces cerevisiae (GRAS), obviating the need for methanol induction, resulting in successful expression. Ultimately, utilizing the GTR-CRISPR editing system, shake flask enzyme activity reached 2 U/ml. The optimal chitosanase activity was achieved at 55℃ and pH 5, with favorable stability between 30 and 50 °C. Following a 2-h catalytic reaction, the product primarily consisted of chitobiose to chitotetraose, predominantly at the chitotriose position, with a slight increase in chitobiose content observed during the later stages of enzymatic hydrolysis. The results affirm the feasibility of heterologous chitosanase expression through Saccharomyces cerevisiae, underscoring its significant industrial potential.</p><h3 data-test=\"abstract-sub-heading\">Graphical Abstract</h3>\n","PeriodicalId":9067,"journal":{"name":"Bioresources and Bioprocessing","volume":"19 1","pages":""},"PeriodicalIF":4.3000,"publicationDate":"2024-02-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Expression of chitosanase from Aspergillus fumigatus chitosanase in Saccharomyces cerevisiae by CRISPR-Cas9 tools\",\"authors\":\"Qingshuai Zhang, Hui Cao\",\"doi\":\"10.1186/s40643-023-00718-4\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Chitooligosaccharides (COS) find numerous applications due to their exceptional properties. Enzymatic hydrolysis of chitosan by chitosanase is considered an advantageous route for COS production. Heterologous expression of chitosanase holds significant promise, yet studies using commonly employed <i>Escherichia coli</i> and <i>Pichia pastoris</i> strains encounter challenges in subsequent handling and industrial scalability. In this investigation, we opted for using the safe yeast strain Saccharomyces cerevisiae (GRAS), obviating the need for methanol induction, resulting in successful expression. Ultimately, utilizing the GTR-CRISPR editing system, shake flask enzyme activity reached 2 U/ml. The optimal chitosanase activity was achieved at 55℃ and pH 5, with favorable stability between 30 and 50 °C. Following a 2-h catalytic reaction, the product primarily consisted of chitobiose to chitotetraose, predominantly at the chitotriose position, with a slight increase in chitobiose content observed during the later stages of enzymatic hydrolysis. The results affirm the feasibility of heterologous chitosanase expression through Saccharomyces cerevisiae, underscoring its significant industrial potential.</p><h3 data-test=\\\"abstract-sub-heading\\\">Graphical Abstract</h3>\\n\",\"PeriodicalId\":9067,\"journal\":{\"name\":\"Bioresources and Bioprocessing\",\"volume\":\"19 1\",\"pages\":\"\"},\"PeriodicalIF\":4.3000,\"publicationDate\":\"2024-02-02\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Bioresources and Bioprocessing\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.1186/s40643-023-00718-4\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Bioresources and Bioprocessing","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1186/s40643-023-00718-4","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Expression of chitosanase from Aspergillus fumigatus chitosanase in Saccharomyces cerevisiae by CRISPR-Cas9 tools
Chitooligosaccharides (COS) find numerous applications due to their exceptional properties. Enzymatic hydrolysis of chitosan by chitosanase is considered an advantageous route for COS production. Heterologous expression of chitosanase holds significant promise, yet studies using commonly employed Escherichia coli and Pichia pastoris strains encounter challenges in subsequent handling and industrial scalability. In this investigation, we opted for using the safe yeast strain Saccharomyces cerevisiae (GRAS), obviating the need for methanol induction, resulting in successful expression. Ultimately, utilizing the GTR-CRISPR editing system, shake flask enzyme activity reached 2 U/ml. The optimal chitosanase activity was achieved at 55℃ and pH 5, with favorable stability between 30 and 50 °C. Following a 2-h catalytic reaction, the product primarily consisted of chitobiose to chitotetraose, predominantly at the chitotriose position, with a slight increase in chitobiose content observed during the later stages of enzymatic hydrolysis. The results affirm the feasibility of heterologous chitosanase expression through Saccharomyces cerevisiae, underscoring its significant industrial potential.
期刊介绍:
Bioresources and Bioprocessing (BIOB) is a peer-reviewed open access journal published under the brand SpringerOpen. BIOB aims at providing an international academic platform for exchanging views on and promoting research to support bioresource development, processing and utilization in a sustainable manner. As an application-oriented research journal, BIOB covers not only the application and management of bioresource technology but also the design and development of bioprocesses that will lead to new and sustainable production processes. BIOB publishes original and review articles on most topics relating to bioresource and bioprocess engineering, including: -Biochemical and microbiological engineering -Biocatalysis and biotransformation -Biosynthesis and metabolic engineering -Bioprocess and biosystems engineering -Bioenergy and biorefinery -Cell culture and biomedical engineering -Food, agricultural and marine biotechnology -Bioseparation and biopurification engineering -Bioremediation and environmental biotechnology